Project 2: Muscular dystrophy clinical trial foundations. Substantial evidence implicates aberrant reactivation of DUX4 expression as the cause of facioscapulohumeral dystrophy (FSHD). Expression profiling of primary myoblasts transduced with DUX4 has identified DUX4 regulated genes, many of which are involved in germline and early stem cell development. Many of these genes are expressed in FSHD muscle but not in muscle from healthy controls, making them ideal biomarker candidates. As FSHD research enters a translational phase, the development of mechanistic, prognostic, and diagnostic biomarkers becomes critical in moving the translational process forward. Aim 1 is a cooperative clinical study for biomarker evaluation in FSHD that will (a) develop FSHD patient registration, assessments, and sample collection in Seattle that parallel those developed in Rochester; (b) develop FSHD MRI assessment capabilities in Rochester that parallel those developed in Seattle; and (c) perform a focused collaborative clinical study to determine the molecular determinants of the MRI findings characteristic of FSHD and to prioritize the best candidate biomarkers for disease activity. Aim 2 will determine whether CD8+ T cells infiltrating FSHD muscle recognize DUX4 induced cancer/testes (C/T) antigens, and will (a) characterize the phenotype, location, and cytokine profile of the T cell/immune cell infiltrate in skeletal muscle in FSHD; (b) expand T cells from FSHD muscle, determine the T cell receptor Vb gene usage and characterize their function; and (c) determine whether T cells derived from FSHD muscle recognize FSHD muscle antigens. These combined aims represent a synergistic effort to evaluate the relationship between tissue biomarkers, histopathology, MRI imaging characteristics, and muscle function, together with a direct characterization of the T cell infiltrate the immune response in the pathophysiology of FSHD.